Regulation of rtt107 recruitment to stalled DNA replication forks by the cullin rtt101 and the rtt109 acetyltransferase.

Department of Biochemistry and Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Ontario M5S 3E1, Canada.

Abstract

RTT107 (ESC4, YHR154W) encodes a BRCA1 C-terminal domain protein that is important for recovery from DNA damage during S phase. Rtt107 is a substrate of the checkpoint kinase Mec1, and it forms complexes with DNA repair enzymes, including the nuclease subunit Slx4, but the role of Rtt107 in the DNA damage response remains unclear. We find that Rtt107 interacts with chromatin when cells are treated with compounds that cause replication forks to arrest. This damage-dependent chromatin binding requires the acetyltransferase Rtt109, but it does not require acetylation of the known Rtt109 target, histone H3-K56. Chromatin binding of Rtt107 also requires the cullin Rtt101, which seems to play a direct role in Rtt107 recruitment, because the two proteins are found in complex with each other. Finally, we provide evidence that Rtt107 is bound at or near stalled replication forks in vivo. Together, these results indicate that Rtt109, Rtt101, and Rtt107, which genetic evidence suggests are functionally related, form a DNA damage response pathway that recruits Rtt107 complexes to damaged or stalled replication forks.

Rtt107 is recruited to chromatin in response to MMS. (A and B) Logarithmically growing cultures expressing the indicated epitope-tagged proteins were treated with 0% (−) or 0.03% (+) MMS for 1 h, before preparation of chromosome spreads. DNA was stained with DAPI (left), and the indicated epitope-tagged proteins were detected with α-VSV (middle). The merged image is shown in the right panel. (C) VSV signal intensity minus background was quantified and expressed relative to the untagged control. The signal intensity was measured for at least 100 nucleoids in each experiment, and the average of two independent experiments is plotted, with error bars spanning 1 SD.

Rtt107 is recruited to chromatin in response to replication fork stalling. (A) Logarithmically growing cultures were treated with 15 μg/ml camptothecin (+CPT) or 150 mM hydroxyurea (+HU) for 1 h, or they were treated with 8.3 μg/ml α factor for 2 h before addition of 8.3 μg/ml α factor and 0.03% MMS for 1 h (α factor + MMS), before preparation of chromosome spreads. DNA was stained with DAPI (left) and VSV-tagged Rtt107 was detected with α-VSV (right). (B) The role of checkpoint pathways in the localization of Rtt107 was analyzed by treating rad53-11 or mec1Δ sml1Δ cells with 0.03% MMS for 1 h, before preparation of chromosome spreads. DNA was stained with DAPI (left) and VSV-tagged Rtt107 was detected with α-VSV (right). (C) VSV signal intensity minus background was quantified and expressed relative to the untagged control. The signal intensity was measured for at least 100 nucleoids in each experiment, and the average of two independent experiments is plotted, with error bars spanning 1 SD. The analysis of MMS-treated cells from C is included for comparison.

Deletion of RTT107, RTT109, RTT101, MMS1, or MMS22 confers similar phenotypes. (A) Ten-fold serial dilutions of the indicated strains were spotted on media containing 0.03% MMS, 150 mM HU, 40 μM CPT, or no drug (YPD), and strains were grown for 2–3 d at 30°C. (B) Spontaneous Rad53 activation was measured in logarithmically growing cells of the indicated strains by using an in situ kinase assay. An autoradiograph of Rad53 is shown, and the amount of autophosphorylation relative to the wild-type control is indicated. (C) The percentage of cells with spontaneous Ddc2 foci was measured for the indicated strains. At least 200 cells were counted in each of two experiments, and the average of the two experiments is plotted with error bars spanning 1 SD. (D) The percent of unbudded and budded cells was measured for the indicated strains. At least 200 cells were counted in each of two experiments, and the average of the two experiments is plotted with error bars spanning 1 SD.

Rtt107 recruitment to chromatin requires RTT109 and RTT101, but not MMS1, MMS22, or SLX4. (A) Logarithmically growing cultures of the indicated strains were treated with 0.03% MMS for 1 h, before preparation of chromosome spreads. DNA was stained with DAPI (left) and VSV-tagged Rtt107 was detected with α-VSV (middle). The merged image is shown in the right panel. (B) VSV signal intensity minus background was quantified and expressed relative to the untagged control. The signal intensity was measured for at least 100 nucleoids in each experiment, and the average of two independent experiments is plotted, with error bars spanning 1 SD. (C) Deletion of RTT109 or RTT101 does not affect Rtt107 expression. Extracts of logarithmically growing cultures of the indicated yeast strains expressing Rtt107-VSV, grown in the presence or absence of MMS, were fractionated by SDS-PAGE, and immunoblots were probed with α-VSV antibodies.

Recruitment of Rtt101 to chromatin requires RTT109 and RTT107. (A) Logarithmically growing cultures were treated with 0.03% MMS for 1 h, before preparation of chromosome spreads. DNA was stained with DAPI (left), and VSV-tagged Rtt101 was detected with α-VSV (middle). The merged image is shown in the right panel. (B) VSV signal intensity minus background was quantified and expressed relative to the untagged control. The signal intensity was measured for at least 100 nucleoids in each experiment, and the average of two independent experiments is plotted, with error bars spanning 1 SD. (C) Deletion of RTT109 or RTT107 does not affect Rtt101 expression. Extracts of logarithmically growing cultures of the indicated yeast strains expressing Rtt101-VSV, grown in the presence or absence of MMS, were fractionated by SDS-PAGE, and immunoblots were probed with α-VSV antibodies. (D) Rtt101 forms a complex with Rtt107. Extracts from cells expressing the indicated tagged proteins were immunoprecipitated with anti-HA antibody. The input and immunoprecipitate fractions were analyzed on immunoblots with anti-HA antibodies to detect HA-Rtt101 and with anti-myc antibodies to detect Rtt107-13myc.

Rtt107 recruitment to chromatin does not require acetylation of H3-K56. (A) Logarithmically growing cultures of the indicated strains were treated with 0.03% MMS for 1 h, before preparation of chromosome spreads. DNA was stained with DAPI (left) and VSV-tagged Rtt107 was detected with α-VSV (middle). The merged image is shown in the right panel. (B) VSV signal intensity minus background was quantified and expressed relative to the untagged control. The signal intensity was measured for at least 100 nucleoids in each experiment, and the average of two independent experiments is plotted, with error bars spanning 1 SD. (C) Deletion of RTT107 suppresses the temperature sensitivity of hst3Δ hst4Δ. Ten-fold serial dilutions of cultures of the indicated strains were spotted onto YPD (top) or SD-Leu (bottom) and incubated at 30°C or 37°C for 3 d.

Rtt107 localizes to origin-proximal regions after replication fork arrest. (A) ChIP-on-chip analysis was performed following synchronous arrest of wild-type (top) or rtt109Δ (bottom panel) cells in early S phase by releasing α factor–arrested cells into 200 mM HU for 2 h at 23°C. After cross-linking and DNA fragmentation, Rtt107 was precipitated, and enrichment of DNA fragments in the Rtt107-bound fraction relative to the unbound fraction is shown along chromosome X. The signal intensity ratio on a log2 scale is shown on the y-axis and the position along the chromosome is shown on the x-axis. Positive signal represents occupancy by Rtt107, and regions where the positive signal is statistically significant () are shown in yellow. Annotated, confirmed replication origins () are indicated by open squares. Potential origins marked by MCM protein binding (; ) are indicated by closed squares. Origins that are active in HU as measured by accumulation of ssDNA () are indicated by open circles. (B) Distribution of Rtt107 near HML and ARS305 in wild type and rtt109Δ. Data from the ChIP-chip experiment in A was analyzed across the indicated chromosome coordinates of chromosome III. Arrows indicate two regions of Rtt107 binding flanking ARS305. The positions of HML and ARS305 are indicated by gray bars. (C) Distribution of Rtt107 near HMR in wild type and rtt109Δ. Data from the ChIP-chip experiment in A was analyzed across the indicated chromosome coordinates of Chromosome III. The position of HMR is indicated by gray bars.